Project Summary/Abstract Neuroimmune activation and signaling contribute to increased ethanol drinking and possibly alcoholism. Preclinical studies have shown that ethanol preference and drinking are modulated by neuroimmune gene expression and that cycles of ethanol exposure lead to long-lasting changes in expression of neuroimmune genes, including neuronal Toll-like receptor 3 (TLR3). These are highly relevant findings given that we have shown that TLR3 expression in post-mortem human alcoholic brains correlates with lifetime ethanol consumption. Therefore it is important to understand the interaction between neuroimmune signaling and ethanol drinking, and the impact of neuroimmune activation on neurobiological changes that can drive escalated ethanol drinking. As such, the focus of this application is on the effects of the TLR3 agonist Polyinosinic:polycytidylic acid (Poly(I:C)) on escalations in operant ethanol self-administration (SA), the underlying neuroimmune and glutamatergic changes (specifically in relation to metabotropic glutamate receptor 2/3; mGluR2/3) in the nucleus accumbens (Acb) and insular cortex (IC), and the role of the IC?Acb circuit. As such, the primary hypothesis of this application is that neuroimmune signaling (i.e., TLR3 activation) induces glutamatergic adaptations in the Acb and IC?Acb circuitry that drive escalations in ethanol SA. Experiments in Aim 1 will examine changes in neuroimmune signaling and glutamatergic targets in Acb and IC in ethanol SA trained rats following Poly(I:C)-TLR3 activation, and parallel functional glutamatergic adaptations in the AcbC in collaboration with Component 5. Experiments in Aim 2 will examine the ability of an mGluR2/3 agonist and antagonist to block and potentiate, respectively, the escalation in ethanol SA following TLR3 activation, and the role of Acb mGluR2/3. Experiments in Aim 3 will utilize a chemogenetic approach by the incorporation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to examine the effects of inhibition and activation of IC?AcbC projections on the escalation in ethanol SA following TLR3 activation. Next, fMRI will be used to assess cortico-accumbal functional connectively as a consequence of ethanol SA history, TLR3 activation, and mGluR2/3 activation in conjunction with the Scientific Core. This proposal tests novel neuroimmune signaling and IC-reward circuits that integrate into, benefit from and contribute to the Alcohol Research Center?s focus on neurocircuit plasticity induced by ethanol. The proposed studies investigate novel mechanisms and circuits associated with the development of alcohol use disorders that could lead to new therapies.